02916nas a2200217   4500008004100000245022100041210006900262260000800331300001000339490000800349520205500357100002002412700002602432700001902458700001502477700002102492700001602513700001802529700002102547856013002568       2017                        eng d00aDifferential rubisco content and photosynthetic efficiency of rol gene integrated Vinca minor transgenic plant: correlating factors associated with morpho-anatomical changes, gene expression and alkaloid productivity0 aDifferential rubisco content and photosynthetic efficiency of ro  cDEC  a12-210 v2193 aTransgenic plants obtained from a hairy root line (PVG) of Vinca minor were characterized in relation to terpenoid indole alkaloids (TIAs) pathway gene expression and vincamine production. The hairy roots formed callus with green nodular protuberances when transferred onto agar-gelled MS medium containing 3.0 mg/l zeatin. These meristematic zones developed into shoot buds on medium with 1.0 mg/l 2, 4-dichlorophenoxyacetic acid and 40 mg/l ascorbic acid. These shoot buds subsequently formed rooted plants when shifted onto a hormone-free MS medium with 6% sucrose. Transgenic nature of the plants was confirmed by the presence of rol genes of the Ri plasmid in them. The transgenic plants (TP) had elongated internodes and a highly proliferating root system. During glass house cultivation TP consistently exhibited slower growth rate, low chlorophyll content (1.02 +/- 0.08 mg/gm fr. wt.), reduced carbon exchange rate (2.67 +/- 0.16 mu mol m(-2) s(-1)), less transpiration rate (2.30 +/- 0.20 mmol m(-2) s(-1)) and poor stomatal conductance (2.21 +/- 0.04 mmol m-2 s(-1)) when compared with non-transgenic population. The activity of rubisco enzyme in the leaves of TP was nearly two folds less in comparison to non-transgenic controls (1.80 milliunits ml(-1) mg protein-1 against 3.61 milliunits ml(-1) mg protein-1, respectively). Anatomically, the TP had a distinct tetarch arrangement of vascular bundles in their stem and roots against a typical ployarched pattern in the non-transgenic plants. Significantly, the transgenic plants accumulated 35% higher amount of total TIAs (3.10 +/- 0.21% dry wt.) along with a 0.03% dry wt. content of its vasodilatory and nootropic alkaloid vincamine in their leaves. Higher productivity of alkaloids in TP was corroborated with more than four (RQ = 4.60 +/- 0.30) and five (RQ = 5.20 +/- 0.70) times over-expression of TIAs pathway genes tryptophan decarboxylase (TDC) and strictosidine synthase (STR) that are responsible for pushing the metabolic flux towards TIAs synthesis in this medicinal herb.1 aVerma, Priyanka1 aKhan, Shamshad, Ahmad1 aMasood, Nusrat1 aManika, N.1 aSharma, Abhishek1 aVerma, Neha1 aLuqman, Suaib1 aMathur, Ajay, K.  uhttps://library.ncl.res.in/content/differential-rubisco-content-and-photosynthetic-efficiency-rol-gene-integrated-vinca-minor02549nas a2200265   4500008004100000022001400041245016000055210006900215260000800284300001400292490000700306520161000313653003601923653001201959653001001971653003101981653001602012100002002028700001802048700002602066700002402092700001802116700001902134856013002153       2020                        eng d  a0971-589400aTIAs pathway genes and associated miRNA identification in Vinca minor: supporting aspidosperma and eburnamine alkaloids linkage via transcriptomic analysis0 aTIAs pathway genes and associated miRNA identification in Vinca   cAUG  a1695-17110 v263 a<p>V. minorcontains monomeric eburnamine-type of indole alkaloids having utilization as a neuro-medicinal plant. The biosynthetic pathway studies using miRNAs has been the focal point for plant genomic research in recent years and this technique is utilized to get an insight into a possible pathway level study inV. minoras understanding of genes in this prized medicinal plant is meagrely understood. The de novo transcriptomic analysis using Illumina Next gen sequencing has been performed in glasshouse shifted plant and transformed roots to elucidate the possible non confirmed steps of terpenoid indole alkaloids (TIAs) pathway inV. minor. A putative TIA pathway is elucidated in the study including twelve possible TIAs biosynthetic genes. The specific miRNA associated with TIAs pathway were identified and their roles were discussed for the first time inV. minor. The comparative analysis of transcriptomic data of glasshouse shifted plant and transformed roots showed that the raw reads of transformed roots were higher (83,740,316) compared to glasshouse shifted plant (67,733,538). The EST-SSR prediction showed the maximum common repeats among glasshouse shifted plant and transformed roots, although small variation was found in trinucleotide repeats restricted to glasshouse shifted plant. The study reveals overall 37 miRNAs which were observed to be true and can have a role in pathway as they can regulate the growth and alkaloid production. The identification of putative pathway genes plays an important role in establishing linkage between Aspidosperma and Eburnamine alkaloids.</p>
10ade novo transcriptomic analysis10aEST-SSR10amiRNA10aTerpenoid indole alkaloids10aVinca minor1 aVerma, Priyanka1 aSingh, Noopur1 aKhan, Shamshad, Ahmad1 aMathur, Ajay, Kumar1 aSharma, Ashok1 aJamal, Farrukh  uhttps://library.ncl.res.in/content/tias-pathway-genes-and-associated-mirna-identification-vinca-minor-supporting-aspidosperma02729nas a2200253   4500008004100000022001400041245018000055210006900235260000800304300001400312490000700326520183900333653001802172653001002190653001502200653000902215653001602224653001402240100002002254700002602274700002402300700002402324856012702348       2021                        eng d  a0971-589400aZCTs knockdown using antisense LNA GapmeR in specialized photomixotrophic cell suspensions of Catharanthus roseus: Rerouting the flux towards mono and dimeric indole alkaloids0 aZCTs knockdown using antisense LNA GapmeR in specialized photomi  cJUL  a1437-14530 v273 aThe present study was carried out to silence the transcription factor genes ZCT1, ZCT2 and ZCT3 via lipofectamine based antisense LNA GapmeRs transfection into the protoplasts of established photomixotrophic cell suspensions. The photomixotrophic cell suspensions with a threshold of 0.5% sucrose were raised and established using two-tiered CO2 providing flasks kept under high light intensity. The photomixotrophic cell suspensions showed morphologically different thick-walled cells under scanning electron microscopic analysis in comparison to the simple thin-walled parenchymatous control cell suspensions. The LC-MS analysis registered the vindoline production (0.0004 +/- 0.0001 mg/g dry wt.) in photomixotrophic cell suspensions which was found to be absent in control cell suspensions. The protoplasts were isolated from the photomixotrophic cell suspensions and subjected to antisense LNA GapmeRs silencing. Three lines, viz. Z1A, Z2C and Z3G were obtained where complete silencing of ZCT1, ZCT2 and ZCT3 genes, respectively, was observed. The Z3G line was found to show maximum production of vindoline (0.038 +/- 0.001 mg/g dry wt.), catharanthine (0.165 +/- 0.008 mg/g dry wt.) and vinblastine (0.0036 +/- 0.0003 mg/g dry wt.). This was supported by the multifold increment in the gene expression of TDC, SLS, STR, SGD, d4h, dat, CrT16H and Crprx. The present work indicates the master regulation of ZCT3 knockdown among all three ZCTs transcription factors in C. roseus to enhance the terpenoid indole alkaloids production. The successful silencing of transcription repressor genes has been achieved in C. roseus plant system by using photomixotrophic cell cultures through GapmeR based silencing. The present study is a step towards metabolic engineering of the TIAs pathway using protoplast transformation in C. roseus.10aCatharanthine10aLC-MS10aProtoplast10aTIAs10aVinblastine10aVindoline1 aVerma, Priyanka1 aKhan, Shamshad, Ahmad1 aParasharami, Varsha1 aMathur, Ajay, Kumar  uhttps://library.ncl.res.in/content/zcts-knockdown-using-antisense-lna-gapmer-specialized-photomixotrophic-cell-suspensions02920nas a2200253   4500008004100000022001400041245010000055210006900155260000800224300001200232520206200244653003102306653002002337653001602357653002802373653001102401653001402412653002502426653001602451100002602467700002002493700002802513856012502541       2022                        eng d  a0167-685700aHomo and heterologous expression of the HpPKS2 gene in Hypericum perforatum and Bacopa monnieri0 aHomo and heterologous expression of the HpPKS2 gene in Hypericum  cJAN  a215-2153 a<p>Hypericum perforatum has been known to produce hypericin and hyperforin that are used in treating mild to moderate depression. The HpPKS2 (H. perforatum polyketide synthase 2) gene is assumed to play a role in hypericin biosynthesis. The HpPKS2 gene was overexpressed in homologous H. perforatum in vitro grown plants through Agrobacterium tumefaciens-mediated genetic transformations. It leads to the establishment of seven glass house acclimatized transgenic lines. Among them, the HP12 transgenic plant showed 9.8 fold enhancement in hypericin content (379.4 +/- 10.3 mu g/g dry wt) followed by 3-fold in HP41 (117.1 +/- 4.5 mu g/g dry wt) as compared to control plants. This was further supported by the real-time PCR studies where it registered up to 5 fold enhancement of HpPKS2 gene expression. On the other hand, the heterologous expression of the HpPKS2 gene in Bacopa monnieri resulted in the establishment of five transgenic plant clones that were successfully acclimatized under glasshouse conditions. Among them, BT4 was found to be very slow-growing. The BT3 line showed maximum expression of the HpPKS2 gene which surprisingly also upregulates the expression of the other metabolic pathway genes of B. monierri namely isopentyl- diphosphate delta isomerase (IDDI), squaline synthase (SQS) and acetyl CoA C acetyltransferase (AA). The HPLC analysis in the heterologous system revealed the maximum production of bacopaside I (9.86 +/- 1.0 mg/g dry wt), bacopaside II (5.89 +/- 0.9 mg/g dry wt) and bacopasaponin C (3.59 +/- 0.3 mg/g dry wt) by transgenic lines BT8, BT3 and BT4, respectively. This enhancement in bacopaside I, bacopaside II and bacopasaponin C production was more than 26-fold, 5-fold and 21-fold, respectively in comparison to the control non transformed plants. Key message Overexpression of the HpPKS2 gene in H. perforatum led to higher hypericin content in the native system while its expression in the heterologous system i.e. B. monnieri also improves bacopaside I, bacopaside II and bacopasaponin C production.</p>
10aAgrobacterium tumefacience10aBacopa monnieri10aBacopasides10aHeterologous expression10aHpPKS210aHypericin10aHypericum perforatum10aTransgenics1 aKhan, Shamshad, Ahmad1 aVerma, Priyanka1 aParasharami, Varsha, A.  uhttps://library.ncl.res.in/content/homo-and-heterologous-expression-hppks2-gene-hypericum-perforatum-and-bacopa-monnieri